Long chain di-carboxylic acids are versatile chemical intermediates that are used in the synthesis of perfumes, polymers, adhesives and lubricants, as well as by the pharmaceutical industry for the synthesis of antibiotics. Diacids with carbon chains longer than C10-C12 carbons, offer potential advantages over shorter diacids but current methods of synthesis of these compounds is commercially inadequate. One of the most important uses of such compounds is in the synthesis of polymers. In these uses long-chain di-carboxylic acids provide greater flexibility and strength than is available with short-chain di-carboxylic acids due to their ability to bend, which minimizes breakage and reduces the number of links in a polymers.
Current methods for the synthesis of long chain dicarboxylic acids involve either the chemical oxidation of unsaturated fatty acids, or the enzymatic oxidation of alkanes or fatty acids using whole cell fermentations (Huf, S. et al. Eur. J Lipid Sci. Technol. (2011), 113: 548-561). Chemical methods for the synthesis of dicarboxylic acids require derivatized fatty acids, such as unsaturated fatty acids (e.g., oleic, palmitoleic), hydroxylated fatty acids (e.g., ricinoleic acid), and the like. Typical chemistries that are used for these transformations include ozonolysis, which oxidizes an alkene to two carboxylic acids. Because ozonolysis is expensive and difficult to practice at the industrial scale, other chemical methods for the oxidation of unsaturated fatty acid have been developed. But these methods often suffer from lower yields (Warwel S, et al Lipid Technol 1997, 9:10-14). Various methods developed for the synthesis of dicarboxylic acids from fatty acids are described in Metzger, J. O., Eur. J. Lipid Sci. Technol. 2009, 111, 865-876. Other chemical methods such as cross metathesis technologies have been reported, but are not currently used in the commercial manufacturing of long fatty acids.
Some biocatalytic approaches for the synthesis of large (>C12) di-carboxylic acids utilize omega hydroxylase in the P450 family. These approaches utilize yeasts such as Candida, which oxidize the alpha and omega positions saturated wax hydrocarbons such as tridecane to tridecanedioic acid (Shuchen, L; et al Enz. Microb. Technol. 2004, 34, 73; Liu, S C; et al Pertochem Technol. 2002, 31, 558). But these methods rely on the use of non-renewable petrochemical waxes and require complicated fermentations of engineered bacteria with limited titers yields and productivities (Huf, S. et al Eur. J Lipid Sci. Technol. 2011, 113, 548). These methods also are limited to di-carboxylic acids having the same number of carbons as the starting wax or fatty acid. (Huf, S et al Eur. J Lipid Sci. Technol. (2011), 113, 548-561).
The P450 enzyme CYP107H1 (Biol) oxidizes fatty acids with C12 to C16 carbons to produce a single product, pimelic acid (heptanedioic acid C7). Besides being very selective for its products, CYP107H1 requires acyl-carrier-protein (ACP)-bound fatty acid as substrate. When free fatty acids are used as substrates, CYP107H1 gives a number of mono-hydroxylation products. Another disadvantage of this enzyme is the requirement for two additional enzymes, a ferredoxin and a feredoxin reductase, that decrease the activity and catalytic turnover of the enzyme (Lawson, R. J et al Biochemistry 2004, 43, 12390; Cryle, M J, Schlichting I. PNAS, 2008, 105, 15696).
It would be useful to have enzymes that could produce dicarboxylic acid products by over-oxidation of the substrates and also perform cleavage of the C—C bonds. This would be particularly useful if it could be done with a single enzyme, and from renewable resources such as fatty acids.